Adjuvant Chemotherapy for Brain Tumors Delivered via a Novel Intra-Cavity Moldable Polymer Matrix
et al. (2013) Adjuvant Chemotherapy for Brain Tumors Delivered via a Novel
Intra-Cavity Moldable Polymer Matrix. PLoS ONE 8(10): e77435. doi:10.1371/journal.pone.0077435
Adjuvant Chemotherapy for Brain Tumors Delivered via a Novel Intra-Cavity Moldable Polymer Matrix
Cheryl V. Rahman 0
Stuart J. Smith 0
Paul S. Morgan 0
Keith A. Langmack 0
Phil A. Clarke 0
Alison A. 0
Donald C. Macarthur 0
Felicity R. Rose 0
Kevin M. Shakesheff 0
Richard G. Grundy 0
Waldemar Debinski, Wake Forest University, School of Medicine, United States of America
0 1 Division of Drug Delivery and Tissue Engineering, School of Pharmacy, University of Nottingham , Nottingham , United Kingdom , 2 Children's Brain Tumor Research Centre, School of Clinical Sciences, University of Nottingham , Nottingham , United Kingdom , 3 Division of Pre-Clinical Oncology, University of Nottingham , Nottingham , United Kingdom , 4 Medical Physics , Nottingham University Hospitals , Nottingham , United Kingdom , 5 Department of Neurosurgery, University Hospital, Queen's Medical Centre , Nottingham , United Kingdom
Introduction: Polymer-based delivery systems offer innovative intra-cavity administration of drugs, with the potential to better target micro-deposits of cancer cells in brain parenchyma beyond the resected cavity. Here we evaluate clinical utility, toxicity and sustained drug release capability of a novel formulation of poly(lactic-co-glycolic acid) (PLGA)/poly(ethylene glycol) (PEG) microparticles. Methods: PLGA/PEG microparticle-based matrices were molded around an ex vivo brain pseudo-resection cavity and analyzed using magnetic resonance imaging and computerized tomography. In vitro toxicity of the polymer was assessed using tumor and endothelial cells and drug release from trichostatin A-, etoposide- and methotrexateloaded matrices was determined. To verify activity of released agents, tumor cells were seeded onto drug-loaded matrices and viability assessed. Results: PLGA/PEG matrices can be molded around a pseudo-resection cavity wall with no polymer-related artifact on clinical scans. The polymer withstands fractionated radiotherapy, with no disruption of microparticle structure. No toxicity was evident when tumor or endothelial cells were grown on control matrices in vitro. Trichostatin A, etoposide and methotrexate were released from the matrices over a 3-4 week period in vitro and etoposide released over 3 days in vivo, with released agents retaining cytotoxic capabilities. PLGA/PEG microparticle-based matrices molded around a resection cavity wall are distinguishable in clinical scanning modalities. Matrices are non-toxic in vitro suggesting good biocompatibility in vivo. Active trichostatin A, etoposide and methotrexate can be incorporated and released gradually from matrices, with radiotherapy unlikely to interfere with release. Conclusion: The PLGA/PEG delivery system offers an innovative intra-cavity approach to chemotherapeutics for improved local control of malignant brain tumors.
Funding: This work was supported by the Joseph Foote Foundation and a Nottingham Advanced Research Fellowship. The funders had no role in study
design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing interests: The authors have declared that no competing interests exist.
Central nervous system (CNS) tumors are the major cause
of cancer related death in children and adults up to the age of
40. This is particularly the case for children with high grade
glioma (HGG), incompletely resected ependymoma (especially
those under 3 years of age) and primitive neuroectodermal
tumors (PNET) who have frequent tumor recurrence and poor
survival rates despite multimodal therapy . Despite the
invasive nature of high grade glioma, most relapses occur
within the wall of the surgical resection cavity, as do most
ependymoma relapses. A subset of HGG (potentially
identifiable on advanced MRI) may display limited invasion and
be particularly amenable to effective localized therapy . In
adults, glioblastoma multiforme (GBM) is the most frequent and
aggressive primary brain tumor and despite radical surgery,
radiotherapy and chemotherapy, median survival after
diagnosis remains only 14 months . Conventional oral or
intravenous chemotherapy distributes drugs to the whole body
whereby toxicity of the drug limits the maximum dose
achievable within the targeted tumor site, particularly for CNS
tumors where the blood brain barrier (BBB) restricts drug influx
from the circulation. There is therefore a need to develop more
effective and targeted chemotherapy regimes that can
eradicate residual brain tumor cells following surgical resection,
thereby improving local control by targeting neoplastic cells
within brain parenchyma beyond the surgical cavity wall and
reducing the risk of tumor recurrence .
Undesired toxicity as a result of systemic administration of
chemotherapeutics and drug doses below the therapeutic
window at the tumor site (due to the BBB) are the driving force
behind the development of local drug delivery systems.
Furthermore, most recurrences of high grade tumors occur
within ~2cm of the wall of the resection cavity. The opportunity
to deliver therapeutic cancer drug concentrations locally
creates the possibility of improving both the safety (low toxic
dose systemically) and efficacy (high effective dose locally) of
cancer chemotherapy, thereby enhancing the benefit of
surgery, as well as continuing anti-neoplastic treatment during
the interval between surgery and commencement of systemic
Drug delivery technologies have developed from simple
tablets, liquids and injectables to sophisticated systems utilizing
bioengineered products. Many of these emerging products use
polymer-based drug delivery systems including microspheres
and nanoparticles, each technology offering unique
characteristics to optimize drug delivery . Such drug delivery
modes may be an important armament for the future treatment
of malignant brain tumors as they encompass strategies to
enhance BBB penetration, to specifically target brain tumor
cells and to implant the polymer at close proximity to residual
cancer cells . For example, OncoGel, a
controlledrelease formulation of paclitaxel in ReGel has shown much
pre-clinical promise. This system comprises a thermosensitive
triblock copolymer (PLGA-PEG-PLGA) which is water soluble
at 2-15C and turns into a viscous gel at body temperature
[8,17]. Pre-clinical and early clinical investigations
demonstrated OncoGels ability to physically target paclitaxel
to esophageal and brain tumor tissue via intralesional injection
into the tumor cavity following resection, with an acceptable
safety profile and moderate increase in survival in a rat
gliosarcoma model [16,18,19]. The rationale of these
polymerbased approaches is to improve upon drug efficacy, increase
exposure time of tumor cells to drug, protect drugs from
degradation and clearance by the immune system until its
release from the polymer, reduce the debilitating sequelae of
current systemic chemotherapeutics and to allow oncological
treatment to be maintained in the interval between surgery and
Although a myriad of drug-polymer devices have been
developed to date, the Food and Drug Administration (FDA)
and National Institute for Health and Clinical Excellence (NICE)
has solely approved the use of chemotherapy impregnated
polymeric wafers (Gliadel) for local chemotherapy delivered
via a biomaterial, for the treatment of primary and recurrent
malignant glioma. These wafers which are neurosurgically
implanted at the time of tumor resection, gradually release the
chemotherapeutic agent carmustine, which then diffuses into
the surrounding brain and targets the residual cancer cells that
have infiltrated the brain tissue. These studies and trials offer
hope to this mode of intra-cavity drug delivery, with results
showing a moderate but significant survival benefit of 2.3
months and 1.8 months median survival for newly diagnosed
and recurrent high grade gliomas respectively .
Nevertheless the treatment has shown limited efficacy mainly
due to: (i) poor drug diffusion, restricted to 2-3mm bordering
the implant; (ii) implants not maintaining close contact with the
resection cavity rim and potentially falling to the bottom of the
cavity; (iii) only one drug being delivered . In the case of
Gliadel the biomaterial was specifically engineered around
the drug carmustine, rather than offer a drug delivery system
widely applicable for the delivery of other chemotherapeutic
Shakesheff and colleagues previously described a novel
temperature-sensitive and biodegradable formulation based on
blended poly(lactic-co-glycolic acid) (PLGA) and poly(ethylene
glycol) (PEG) microparticles . This technology has
successfully been used to deliver an oesteogenic growth factor
for bone repair in a murine calvarial defect model . The
microparticles are a free-flowing powder at room temperature
and create a paste when mixed with a saline-based carrier
solution. The formulation can be injected or pasted at room
temperature and then gradually solidifies (sinters) into a solid,
porous matrix at body temperature. Multiple chemotherapeutic
agents can be loaded into the carrier solution phase that is
mixed with the microparticles to form a paste. The
chemotherapeutic agents adsorb to the surface of the
microparticles and absorb into the microparticle bulk structure.
This paste can then be molded around the resection cavity
wall. Within approximately 15 minutes at body temperature, the
particles solidify into a porous matrix which then gradually
releases the chemotherapy. Solidification only at body
temperature (a process which is not exothermic) aids retention
of the drug-loaded matrix within the cavity wall. PLGA polymer
degrades via hydrolysis over a period of months and is
removed from the body through excretion of the lactic and
glycolic acid by-products via metabolic pathways such as the
tricarboxylic acid cycle . PLGA was chosen because it has
a long history of use as a FDA-approved degradable medical
implant material with well established safety in the clinic (for
example low cytotoxicity and good biocompatibility of
resorbable sutures) . Importantly, PLGA microspheres
gradually degrade and have good biocompatibility to brain
tissue [34,35]. The properties of the PLGA/PEG microparticle
based-matrices potentially offer several advantages over
systemic administration and current polymeric delivery
systems: (i) matrices can be molded into any size or shape
desired, such as the irregular surgical resection cavity wall; (ii)
capability to incorporate and release multiple drugs increasing
the flexibility of treatment offered to the clinician and patient;
(iii) sustained drug release over 1-3 weeks providing
oncological treatment in the interval before commencement of
Here we demonstrate the novel mode of application of the
PLGA/PEG microparticle-based matrix drug delivery system for
brain tumors for which complete surgical resection is not
achievable. We describe its clinical compatibility with respect to
radiotherapy and magnetic resonance imaging/computerized
tomography (MRI/CT) scanning modalities. Our
laboratorybased studies evaluate in vitro sustained release of trichostatin
A (TSA), etoposide (ETOP) and methotrexate (MTX) from
PLGA/PEG microparticle-based matrices, short-term in vivo
release of etoposide and assess whether released agents
retain cytotoxic function.
Materials and Methods
PLGA/PEG particle production
Thermosensitive particles were fabricated from blends of
53kDa PDLLGA (85:15 DLG 4CA) (Lakeshore Biomaterials,
USA) and PEG 400 (Sigma Aldrich, UK) as previously
described . Briefly, a mixture of 93.5%:6.5% PLGA:PEG
(w/v) was blended at 80-90C on a hotplate. The melted PLGA
and PEG were mixed together by hand using a PTFE-coated
spatula and allowed to cool. Polymer blend sheets were then
ground into particles in a bench-top mill (Krups Mill F203) and
the particles were sieved to obtain the 100-200m particle size
Composite matrices were prepared in PTFE moulds. For in
vitro release experiments cylindrical scaffolds of 12mm length
and 6mm diameter were produced. The PLGA/PEG particles
were mixed manually with drug solution (trichostatin A,
etoposide or methotrexate) or water (negative controls). A ratio
of 1:0.6 of particles to solution was used. The drug loaded
matrices contained 50g of drug per matrix. The particle paste
was then packed into the mould which was placed at 37C for 2
hours to allow matrix formation (sintering). For in vitro
biocompatibility and toxicity assays cylindrical scaffolds 1mm
thick and 4mm diameter were produced. The PLGA/PEG
particles were mixed manually with drug solution (trichostatin
A, etoposide or methotrexate) at a ratio of 1:0.6 of particles to
solution. The drug loaded matrices contained 50g of drug per
matrix. The particle paste was then packed into the mould
which was placed at 37C for 2 hours to allow matrix formation.
Experimental cell lines and culture
PFSK-1 (childhood central nervous system primitive
neuroectodermal tumor) (CNS PNET)), DAOY (childhood
medulloblastoma), U87 (adult glioblastoma multiforme) and C6
(rat glioma) cell lines were previously characterized by and
purchased from ATCC. Monolayer cells were cultured in
DMEM (Sigma, UK), supplemented with 10% fetal bovine
serum (PAA Labs, UK), 5mM sodium pyruvate, 5mM
LGlutamine and maintained in a humidified incubator at 37C
and 5% CO2. To assess in vitro biocompatibility and toxicity of
PLGA/PEG matrices cells were seeded in triplicate. Matrices
were placed in single wells of a 24-well plate and pre-treated
with 50l of culture media. A 30l suspension containing 1x105
cells were placed onto each matrix and plates incubated for 2
hours at 37C to allow cells to adhere to the polymeric
microparticles. Fresh culture media (1ml) was added to each
well ensuring matrices were submerged, and incubated at 37C
until required for assaying.
Ex vivo application of PLGA/PEG matrices
Sheep heads were obtained from a local abattoir after
permission was granted to use these animal parts (C Brumpton
Butchers Ltd, Nottingham). Heads were fixed in a vice and
bilateral skin and muscle flaps raised. Three burrholes were
performed with a Hudson brace on each fronto-temporal region
of the cranium and joined with a Gigli saw to raise
craniotomies. The dura was incised and flapped back, and
secured with vicryl stay sutures. Incisions were made through
the pia and the brain parenchyma excised to give a cavity of
dimensions 2.5x2.5x2.5cm. PLGA/PEG blended microparticles
were mixed with PBS in a 1:0.6 ratio to form polymer-based
paste and applied to the ovine pseudo-resection cavity.
Cavities were either filled completely with PLGA/PEG or lined
all around to an approximate depth of 2mm.
MRI and CT scanning of ex vivo brain
MR imaging was performed using a clinical 3 T Achieva MR
scanner (Philips Medical, Best, The Netherlands) with the
specimen placed inside the 8 channel receive-only head coil.
Routine fat-suppressed 3D turbo spin echo T1- and
T2weighted whole brain imaging was performed with acquisition
resolution of 1x1x1 mm, 192x192x160 matrix, echo train length
of 133, echo time of 262 ms, repetition time of 2500 ms, and
464 s overall scan time. The CT scans were acquired using an
adult head protocol on a clinical Mx8000 IDT 16 scanner
(Philips Medical, Best, The Netherlands). 144 slices, 1.5 mm
thick, were acquired at 120 kV with 512x512 matrix and 0.45
mm in-pixel pixel size.
Chemotherapeutic and experimental agents
TSA and ETOP were purchased from Sigma, UK and
resuspended in dimethyl sulfoxide (DMSO) to give 5mM and
50mM working concentrations respectively. MTX was
purchased from Sigma, UK and resuspended in phosphate
buffered saline to give a 100mg/ml stock solution.
Radiotherapy dosing regime
A 12mm x 6mm sample of the biomaterial was placed within
a cavity of the head region of an anthropomorphic phantom
(RandoTM phantom, The Phantom Laboratory, Salem, NY,
USA). The head area was then CT scanned to give a set of
contiguous 3 mm slices (Aquilion LB CT scanner, Toshiba,
Crawley, UK). These CTs were transferred to a radiotherapy
treatment planning system (Oncentra version 3.3 SP3,
Nucletron, UK) using a DICOM protocol. Within the planning
system the sample material was outlined and a region of
interest 1 cm in all directions was grown to provide a target
volume. An experienced radiotherapy planner then devised a
treatment plan to irradiate the target volume as uniformly as
possible to 60 Gy using a 6 MV beam. A standard isocentric 3
field arrangement was used, which gave a mean target dose of
60 Gy (range 59.3 Gy to 60.7 Gy). The plan was delivered as
30 daily 2 Gy fractions using a clinical linear accelerator (Elekta
Precise, Elekta, Crawley, UK). The fractionation schedule was
as routine clinical practice, i.e. one fraction per day Monday to
Friday, no irradiations at the weekends.
Live/Dead viability assay
Brain tumor cells were seeded onto PLGA/PEG matrices as
described previously and Live/Dead viability/cytotoxicity
assay (Invitrogen, UK) conducted on days 1,2 and 3
postseeding. 5l of 2mM ethidium homodimer-1 and 2.5l of 4mM
calcein AM were added to 10ml DMEM and 1ml of the final
solution was used to stain and completely submerge one
cellseeded PLGA/PEG matrix (one matrix per well of 24-well
plate). Following incubation for one hour at 37C, matrices
were rinsed three times with DMEM to remove background
staining and a further three times with PBS. Each matrix was
carefully placed on a microscope slide and fluorescence
signals (green = viable; red = dead) visualized using a Leica
DMRB upright fluorescent microscope. Matrices were placed
back into culture to ensure tracking of the cells present on the
same matrix over 3 days with respect to viability observations
and quantitation of fluorescent cells. Three matrices were used
for each cell line and 100 cells were counted for each matrix to
determine the proportion of live:dead cells.
Alamar Blue proliferation assay
Brain tumor cells were seeded onto PLGA/PEG matrices in
24-well plates as described above. On days 1, 2 and 3
postseeding, matrices were washed three times with warm PBS
and finally placed in 1ml PBS. 100l Alamar Blue indicator dye
(Invitrogen, UK) was added to each well ensuring matrices
were submerged. Following incubation for 90 minutes at 37C,
100l aliquots were transferred to single wells of a 96-well
black-bottom plate in triplicate and fluorescence emission
measured at 585nm using a plate reader (Tecan, Switzerland).
Three matrices were used for each cell line and fluorescent
readings were measured for each matrix in triplicate. Matrices
were placed back into a single well of a 24-well plate and fresh
culture media added to ensure tracking of the same matrix over
3 days with respect to cell proliferation. Fluorescence intensity
over a 3-day culture period was compared to counterpart
monolayer cells. Data is presented as the average relative
fluorescence units of three independent experiments with error
bars indicating standard error of the mean values.
Scanning electron microscopy
Cell-seeded PLGA/PEG matrices were washed three times
in fresh PBS and fixed in 3% glutaraldehyde for 12 hours.
Fixed samples were then washed three times in PBS and
dehydrated for a further 12 hours by exposure to air at room
temperature. Fixed and dehydrated cell-seeded matrices were
mounted on aluminium stubs and sputter-coated with gold at
an argon current rate of 30mA for 3 minutes. Cell attachment
and morphology on the matrices was visualized using a
scanning electron microscope (SEM) (JEOL JSM-6060LV) at
In vitro drug release from PLGA/PEG matrices
50g or 100g drug-loaded matrices were placed in 4ml
fresh PBS buffer pH 7.4 and incubated at 37C. At various time
intervals, the entire volume of PBS was replaced with 4ml fresh
release buffer and 100l of buffer containing released drug was
sampled in triplicate at a relevant UV wavelength (TSA, 342nm,
ETOP, 295nm and MTX, 324nm) and concentration of drug
measured using a standard curve determined at the same time.
Three matrices were used for each drug and blank PBS loaded
PLGA/PEG matrices were used as a control for background
absorbance values. Assays were terminated when 80% or
more of the drug had been released and when no drug release
was detected for two days or more. Data is presented as
cumulative drug release as a function of time.
Cytotoxicity of released agents
50g drug-loaded matrices were placed in 2ml of culture
media in single wells of a 24-well plate. After either 24 hours or
14 days, media was removed and brain tumor cells were
seeded onto matrices using a 30l suspension containing
1x105 cells. Cells were allowed to adhere onto matrices for two
hours prior to adding 2ml fresh culture media to the wells.
Alamar Blue proliferation assay (Invitrogen, UK) was conducted
72 hours post-cell seeding and percentage viability of cells
relative to untreated cells was measured. Data is presented as
the average relative fluorescence units of three independent
experiments with error bars indicating standard error of the
In vivo etoposide release from PLGA/PEG matrices
This study was approved by the University of Nottingham
local Ethical Review Committee and granted by the UK Home
Office (License No. PPL 40/3559), after consideration of the
justification of animal research and good animal welfare. Six
4-6 week old male MF-1 nude mice (3 mice per arm) were
maintained under standard conditions as detailed in the UK
Home Office Animals (Scientific Procedures) Act 1986 and
studies conducted and reported in compliance with the 2010
NC3R ARRIVE guidelines. Animals U87 GBM cells tagged with
a bioluminescent marker (DLuX) were injected subcutaneously
into the left flank and the tumour grown for 15 days whilst
monitoring using the IVIS Spectrum bioluminescent imaging
system (PerkinElmer, UK). Mice with satisfactory tumour take
and growth rates underwent partial tumour resection. The
previous flank incision was re-opened and a biopsy punch/fine
suction tip used to resect tumour back to the tumour/tissue
interface, thus mimicking the surgical technique utilized in
human patients undergoing comparable surgery for GBM.
Etoposide-loaded PLGA/PEG matrices (experimental arm) or
blank PLGA/PEG matrices (control arm) were moulded around
the resection cavity. Animals were weighed daily by an
experienced technician, any adverse effects noted, and
sacrificed using cervical dislocation once their clinical condition
deteriorated, in order to ameliorate suffering. Mice were
sacrificed 3 days post-implantation and tumour tissue
sectioned prior to staining with hematoxylin and eosin or glial
fibrillary acidic protein (GFAP) (anti-GFAP (Abcam, ab726;
Pearsons correlation coefficient was used to determine the
concordance of drug release rates between PLGA/PEG
formulations with two different drug concentrations. Unpaired
student t-test was used to determine whether proliferation rates
of cells cultured on matrices differed significantly from
corresponding cells cultured as monolayers with a p-value <
0.05 deemed statistically significant. Statistical analyses were
conducted using SPSS software version 6.
PLGA/PEG microparticle-based matrix can be molded
around a resection cavity wall
The PLGA/PEG paste was easily applied to and readily
molded around, an irregular-shaped pseudo-resection cavity
wall ex vivo (Figure 1a and Movie S1). The polymer formulation
fully maintained its shape after 15 minutes sintering at 37C,
with close apposition to the cavity wall (Figure 1b and Movie
PLGA/PEG microparticle-based matrix can be
distinguished by MRI and CT scanning
To establish whether PLGA/PEG interferes with MRI- and
CT-based brain scans by causing image artifacts that obscure
visualization of brain parenchyma and thereby potentially
hampering identification of a recurrent tumor, an ovine head
containing one polymer-filled and one polymer-lined
pseudoresection cavity was scanned ex vivo under standard clinical
procedure. Cavities filled and lined with PLGA/PEG are
distinguishable from the surrounding brain parenchyma using
standard CT scanning (Figure 1 c-d). Similarly, cavities filled
with PLGA/PEG are distinguishable from surrounding brain
parenchyma using T2- and T1- weighted MRI scans (Figure 1
e-f respectively). Edges of the cavity are clearly defined with no
additional image artifacts observed from the PLGA/PEG,
indicating insignificant spatial distortion and good contrast with
brain parenchyma on T2 and T1 MR (Figure 1c). Therefore
application of PLGA/PEG matrices does not interfere with
clinical scanning modalities used as standard procedures for
the detection of brain tumors.
Radiotherapy does not alter PLGA/PEG matrix
In anticipation of radiotherapy treatment for patients with
PLGA/PEG intra-cavity implants, it is important to determine
effects of radiation on the biomaterial and subsequent drug
release. Moreover the effect of radiation on the microstructure
of the PLGA/PEG matrix is unknown. PLGA/PEG matrices,
placed in the brain area of an anthropomorphic phantom were
subjected to a standard isocentric high dose radiotherapy
regime of 60 Gy (2 Gy fractions daily for 30 days) (Figures
2ab). Irradiated matrices show no visible difference in terms of
microparticle size, morphology and distribution throughout the
matrices when compared to control non-irradiated matrices
stored under similar conditions. Microparticles appear
PLGA/PEG matrices are non-toxic to normal and
To address whether PLGA/PEG matrices exert cytotoxicity,
PFSK-1, DAOY, C6 and U87 brain tumor cell lines were
seeded directly onto matrices and cultured for three days. All
seeded tumor cells show distinct 3D morphology both on and
between polymer microparticles, with visible extracellular
matrix laid on the particle surface (Figures 3a-e). The Live/
Dead assay confirmed that ~85% of tumor cells seeded onto
matrices remain viable after three days (Figure 3f-j). When
regarding a three day culture period, there is no significant
difference in metabolic activity of tumor cells grown on matrices
compared to the same cell lines cultured as 2D monolayers
(PFSK-1 p<0.8, DAOY p<0.4, C6 p<0.2 and U87 p<0.6), thus
indirectly demonstrating a comparable proliferation rate over a
three day culture period (Figure 3k-n). To verify that the
observed lack of cellular toxicity due to PLGA/PEG matrices is
Figure 2. Effect of clinically-relevant radiotherapy regime on PLGA/PEG microparticle-based matrices. (A) PLGA/PEG
matrices (12mm x 6mm) were placed within cavities in the brain area of an anthropomorphic phantom. (B) A head computerized
tomography was obtained (3mm slices) using a standard radiotherapy protocol and the computerized tomography transferred to a
radiotherapy planning system to give a uniform standard isocentric three field 60 Gy dose (30 daily 2 Gy fractions) of radiation to the
biomaterial and surrounding area. (C) Scanning electron microscopy of control non-irradiated matrices kept at room temperature for
30 days, showing distinct and structurally intact microparticles with visible pores between particles. (D) Scanning electron
microscopy of irradiated matrices showing no obvious difference in matrix microstructure (scale bar 100m).
not observed exclusively in cancerous cells, normal brain
endothelial cells (HBMEC) were seeded onto matrices. Over a
three day culture period, endothelial cells were also observed
to proliferate with 100% viability (Figure 3o-p). Thus these
results demonstrate that PLGA/PEG microparticle-based
matrices are non-toxic to neoplastic and normal brain cells.
PLGA/PEG matrices permit sustained release of
chemotherapeutic agents in vitro
To evaluate the capability of PLGA/PEG matrices to release
standard of care and experimental chemotherapeutic agents,
TSA, ETOP and MTX were loaded onto polymer matrices and
drug release profiles measured in vitro. Release of TSA, ETOP
and MTX followed a similar profile, exhibiting an initial burst
phase followed by slower, sustained release profiles (Figure
4a-c). An initial burst release of 44% TSA was observed,
caused by drug on the surface of the scaffolds being released
into the PBS immediately. This was followed by an average
daily release of 1.2% until day 18 (Figure 4, top). At this point
the PLGA/PEG matrices stopped releasing drug, with a total of
94% TSA released. ETOP-loaded matrices exhibited an initial
burst of 29% total drug loaded followed by an average daily
release of 1.3% (Figure 4, middle). Drug release from the
matrices stopped at day 26, with a total of 77% ETOP
released. A burst release of 49% MTX was observed followed
by an average of 0.7% release per day (Figure 4, bottom). The
matrices stopped releasing MTX by day 28, at which point 78%
total loaded MTX was released.
To determine whether the amount of drug loaded influences
the release profile, TSA was loaded onto PLGA/PEG matrices
at 50g and 100g and drug release profiles directly
compared. The initial burst on Day 0 is greater in matrices
loaded with 100g TSA (50%) compared to matrices loaded
with 50g TSA (28%); the release profiles thereafter however,
are comparable with a high degree of correlation over an 8-day
period (Pearsons correlation coefficient, 0.94). Therefore
although the initial burst release of TSA is directly proportional
to the amount of drug loaded, the slower sustained zero-order
release is independent of the amount of drug loaded (Figure
Released agents from PLGA/PEG matrices retain
cytotoxic capabilities in vitro
To determine whether drugs released from PLGA/PEG
microparticle-based matrices retain cytotoxic function, brain
tumor cells were assessed for viability after seeding onto
drugloaded matrices 24 hours after drugs were incorporated and
exposing to released drugs for 72 hours. PFSK-1 is sensitive to
all three agents with approximately 20-70% viable cells
remaining with drug potency in the order: TSA>ETOP>MTX
(Figure 5a). DAOY is sensitive to all three agents with
approximately 30-55% viable cells remaining with drug potency
in the order: TSA/ETOP>MTX (Figure 5b). C6 is sensitive to all
three agents with approximately 20-70% viable cells remaining
with drug potency in the order: TSA>ETOP>MTX (Figure 5c).
U87 is sensitive to TSA and ETOP with approximately 25-75%
viable cells remaining with drug potency in the order:
TSA>ETOP. U87 is insensitive to MTX under the experimental
conditions described here (Figure 5d). Additionally, TSA
cytotoxicity on brain tumor cells was assessed following a 2
week release period in vitro, to demonstrate that the released
drug retains its activity and is capable of exerting cytotoxic
effects after a prolonged period of time within the PLGA/PEG
matrix. TSA-loaded PLGA/PEG matrices were placed in PBS
for 14 days to allow drug release. On day 14 brain tumor cells
were seeded onto matrices and assessed for cell viability after
three days of culture. The results indicate that cumulative
release of TSA on days 15-17 post drug-loading (~4% of total
drug loaded), retains cytotoxic function as cell proliferation is
impaired in all four cell lines investigated at IC50 concentrations
Figure 3. Assessment of PLGA/PEG microparticle-based matrix toxicity to normal and cancerous cells. To address toxicity
of polymer matrices, brain cancer cells were grown on PLGA/PEG matrices. (A) Scanning electron microscopy images of blank
PLGA/PEG matrices with distinct microparticles (m) and pores (p). (B-E) PFSK-1 (CNS PNET), DAOY (medulloblastoma), C6 (rat
glioma) and U87 (glioblastoma multiforme) brain tumor cells cultured on PLGA/PEG matrices for 3 days. Cells exhibit 3D
morphology both across microparticle faces and between microparticles, with extra cellular matrix visible. Representative tumor
cells are denoted with an asterisk. Magnifications x1000. (F) Live/Dead assay of PFSK-1 cells demonstrates that ~85% of cells are
viable. (G) Image of PFSK-1 cells without adjustment of background fluorescence, with visible microparticles (background
fluorescence from particles is reddish-brown). (H-J) Live/Dead assay of DAOY, C6 and U87 cells demonstrates that 85%-100% of
cells are viable. All images are shown on culture day 3 post-seeding onto matrices. Green = viable; Red = non-viable. Scale bar =
100m. (K-N) Alamar Blue proliferation assay for PFSK-1, DAOY, C6 and U87 respectively, over a 3-day culture period
postseeding onto PLGA/PEG matrices and compared to 2D monolayer cultures of the same cell lines. The proportion of proliferating
cells was inferred indirectly from a measurement of metabolic activity. Error bars represent the standard error of the mean from
three independent matrices and over three independent experiments. Unpaired t-test analyses reveals no significant differences
between the two culture systems (O-P) Viability and proliferation of brain endothelial cells (HBMEC) over a 3-day culture period
post-seeding onto PLGA/PEG matrices Green = viable; Red = non-viable. Scale bar = 100m.
Figure 4. In vitro cumulative release profiles of agents loaded onto PLGA/PEG matrices. All drug release behaviors from the
drug-loaded polymer matrices were investigated under simulated physiological conditions (PBS, pH 7.4) at 37C. (Top) TSA exhibits
an initial burst of 44% of total drug loaded with a total of 94% of drug released by day 18 and no drug detected thereafter. (Middle)
ETOP exhibits an initial burst of 29% of total drug loaded with 77% of drug released by day 26. (Bottom) MTX exhibits an initial burst
of 49% of total drug loaded with 78% of drug released by day 28. No drug release is detected thereafter. Three independent
matrices were used for each experimental set-up with 50g of drug loaded per matrix. Day 0 initial burst readings were taken 4h
after drug-loaded matrices were placed in PBS. UV absorbance wavelengths were as follows: TSA, 342nm; ETOP, 295nm; MTX,
324nm. Error bars represent standard error of the mean from three independent matrices.
(Figure S2) These results indicate that chemical and physical
interaction of TSA, ETOP and MTX with the PLGA/PEG
polymer does not impair molecular structure integrity of the
drug, as these agents retain cytotoxic function in vitro.
PLGA/PEG matrices release active etoposide in vivo
Etoposide-loaded polymer was moulded around a flank GBM
resection cavity after performing partial tumour resection. Mice
were sacrificed 3 days after implantation of the drug-loaded
polymer to demonstrate proof-of-concept for in vivo drug
release and gauge short-term drug diffusion distance.
Haematoxylin and eosin staining shows a clear kill-zone visible
beyond the resection boundary consisting of necrotic tumour
cells, demonstrating in vivo release of cytotoxic levels of
etoposide from the polymer matrix. Etoposide drug diffusion
after release from the polymer matrices was approximately
1mm after 3 days, based indirectly upon the region of the
killzone (Figure 6).
The BBB acts as a block to the administration of many
chemotherapeutic agents to the central nervous system. Drugs
have to be given at escalating doses to achieve effective doses
at the tumor site, but this increased dosing leads to significant
side-effects and toxicities systemically, e.g. bone marrow
suppression, nausea or epithelial damage. By administering
chemotherapy directly into the brain tumor cavity, thus
bypassing the BBB, high local doses can be achieved to
ensure maximal anti-cancer activity, whilst minimizing systemic
toxicity. The proof of concept of such an approach has been
achieved by Gliadel; however despite its potential, treatment
with Gliadel has been variable in efficacy due to drug
resistance in patients  and the technical product
characteristics of Gliadel. The rate of release from Gliadel is
also essentially uncontrolled with the majority of drug release
occurring within a few days of implantation, reducing the
chronicity of effect (both in vitro release in saline buffer at 37C
and in vivo studies document a release period of 5 days) and
compounding its limited success in the clinic .
Here we document a PLGA/PEG microparticle-based matrix
that provides a novel application mode of a biomaterial in close
proximity to the tumor bed and micro-deposits of neoplastic
cells directly beyond. When mixed with saline, the PLGA/PEG
microparticle formulation is a paste onto which
chemotherapeutics can be loaded. This paste is easily molded
around a tumor resection cavity after surgery and begins to
solidify into a matrix at body temperature, adhering to the
surgical cavity wall. Close tissue apposition of the PLGA/PEG
matrix to the cavity wall as observed when applied surgically ex
vivo, refines and maximizes the potential of this therapeutic
modality. The ability to mould the local chemotherapy source
around the entire irregular shaped resection cavity offers vastly
superior tissue approximation when compared to discrete
polymeric wafers or polymer-based hydrogels. To our
knowledge, this is the first report of polymer-based drug
Figure 5. Cytotoxicity of agents released from PLGA/PEG matrices. Brain tumor cells were assessed for viability after seeding
onto drug-loaded matrices one day after drugs were loaded, to determine cytotoxic capabilities of released agents. (A-C) PFSK-1,
DAOY and C6 cells are sensitive to all three agents with approximately 20-70% viable cells remaining (D) U87 is sensitive to TSA
and ETOP with approximately 25-75% viable cells remaining but is insensitive to MTX. Percentage cell viability was calculated
relative to PBS-loaded control matrices for each cell line.
delivery system which can be applied as a paste directly onto
the brain surface and molded to the shape of that topography.
It is imperative that the clinical utility of the PLGA/PEG matrix
be evaluated at this proof-of-concept stage prior to
advancement to in vivo studies and early phase patient trials.
Our findings indicate that the PLGA/PEG polymer does not
interfere with MRI and CT scanning of the brain using standard
clinical sequences. No polymer-related artifact was evident in
either scan modality, with biomaterial clearly distinguishable
from brain parenchyma. Therefore it is unlikely that the
presence of the polymer lining a resected cavity would impair
visualization of tumor recurrence in a patient following MRI or
CT scanning. Furthermore, a typical high dose fractionated
radiotherapy course delivered to a patient with intra-cavity
PLGA/PEG matrices, is unlikely to affect the sustained and
gradual release of chemotherapy from the polymer, as no
alteration in the microstructure of the matrix was observed
using an anthropomorphic dummy phantom. Specifically, the
porosity of the microparticles did not appear to differ from
nonirradiated polymer, implying that high dose radiation is unlikely
to cause the remaining drug to be released in a rapid burst due
to the loss of structural integrity of the polymer.
Exposing brain tumor cells and brain endothelial cells to
PLGA/PEG matrices by seeding cells directly onto matrices
allows an indication of whether the polymer formulation is toxic.
Our findings comprehensively show that both neoplastic and
untransformed cells are viable and proliferate during at least a
3-day culture period. This is consistent with the documented
low cytotoxicity and good biocompatibility of resorbable
FDAapproved PLGA medical sutures [31,32]. Similarly, a recent
study using rats with C6 glioma cells orthotopically implanted,
observed no significant difference in survival time between
blank PLGA microparticles interstitially delivered and untreated
control animals, suggesting that PLGA had no inhibitory effect
on C6 cells . Our results suggest that our specific
formulation of PLGA/PEG does not result in cellular toxicity;
however biocompatibility assessment of the PLGA/PEG matrix
with host tissue using in vivo models and during patient trials
will be required to validate these in vitro findings.
Pre-clinical polymer-based drug delivery systems targeting
localized brain tumor chemotherapy have predominantly
utilized single-agent drug release strategies or those involving
only one class of drug (e.g. hydrophobic, lipophilic etc.)
[12,15,3841]. Many of these studies, including those
investigating the in vivo pharmacokinetics of the carmustine
implant (Gliadel), have achieved only up to seven days drug
release at which point carmustine release was almost complete
[25,42]. We have demonstrated 18-28 days sustained drug
release using two standard of care chemotherapeutic agents
and one experimental anti-cancer agent shown by us
previously to exert anti-tumor effects on childhood brain tumor
cells in vitro . These drugs are grouped into two distinct
drug classes: TSA, lipophilic; ETOP and MTX, hydrophobic.
Release profiles of TSA, ETOP and MTX from PLGA/PEG
matrices generally follow a biphasic profile, at least for the
duration of the described experiments . Our principal
outcome measure in this objective was to achieve 3-4 weeks
drug release as this typically represents the lag period between
surgical resection and commencement of standard
chemotherapy administration. Therefore this would potentially
permit oncological (local) treatment as an adjuvant therapy
prior to standard systemic administration. A characteristic initial
burst of between ~30-50% is released rapidly and corresponds
to drug molecules bound to the periphery of the
microparticlebased matrix and attached to the surface of the microparticles.
A sustained release phase follows where 0.7-1.3% of drug is
released per day and corresponds to drug trapped within the
pores between microparticles. Release in this second scenario
occurs via diffusion through the pores. It is likely that an
additional phase would occur upon full degradation of the
PLGA/PEG polymer, at which point the remainder of the drug
would be anticipated to be released. Although we observed a
cessation of MTX release from day 4 to 7, drug release
thereafter continued again from day 8 and continued at a low
level until ~80% of the loaded drug was released from the
matrices. This cessation of MTX release is consistent with a
recent report using chitosan-based nanoparticles for MTX
delivery to brain tumors, which documents a 3-day release
profile with ~80% of MTX still entrapped within the
nanoparticles . In contrast, almost all of TSA is released
from PLGA/PEG matrices in vitro. Our result shows sustained
release of TSA over 18 days, whereas a recent study using
liposomes loaded with TSA reports 100% drug release over 24
hours . Similarly, our results show a more sustained
release profile of ETOP over 24 days, compared to a recent
report using poly(ether-anhydride) particles, which releases
ETOP in vitro over 6 days . The in vitro drug release assay
described in this study provides a screening method for
assessing drug suitability with this delivery system. Importantly,
the physicochemical interactions between these
chemotherapeutics and PLGA do not impair the drug activity as
demonstrated by the capability of drugs released from
PLGA/PEG matrices to exert a cytotoxic effect on brain tumor
cells. Moreover, short-term in vivo studies demonstrate
proofof-concept for drug release within a tumor tissue
microenvironment and approximately 1mm drug diffusion
distance. Longer-term in vivo studies will be required to
corroborate this finding and to determine maximal drug
In summary, we describe a novel intra-cavity drug delivery
system using a PLGA/PEG microparticle-based matrix, which
can be easily pasted around the resected tumor bed. Multiple
chemotherapeutic agents can be loaded onto this polymer
which solidifies at body temperature, gradually releasing the
drugs over time. The PLGA/PEG matrix is amenable to use
with current standard clinical procedures as polymer can be
distinguished during MRI/CT scanning and radiotherapy dosing
does not adversely affect the polymer structure. The polymer is
non-toxic to tumor and normal cells and the chemotherapeutic
agents TSA, ETOP and MTX can be released from matrices in
a sustained manner over 3-4 weeks in vitro with retention of
cytotoxic capability. Our results should expedite in vivo studies
of efficacy and neurotoxicity using combinations of standard of
care agents and ultimately lead to early phase patient trials for
the treatment of high grade brain tumors. Although described
here for the treatment of childhood and adult brain tumors, this
system has applicability to any solid tumor for which complete
surgical resection of the tumor is not achievable.
Figure S2. Cytotoxicity of TSA released in vitro between
days 15-17. Brain tumor cells were seeded onto TSA-loaded
PLGA/PEG microparticle-based matrices 14 days after drugs
were loaded and assessed for viability after 72h. Cumulative
TSA release between days 15-17 post drug-loading retains
cytotoxic capability as proliferation is impaired in PFSK-1,
DAOY, C6 and U87 brain tumor cells. Brain tumor cells seeded
onto PBS-loaded matrices were used as controls.
The authors thank Christine Grainger-Boultby for assistance
with scanning electron microscopy.
Conceived and designed the experiments: RR CVR SJS.
Performed the experiments: RR CVR SJS PSM KAL PAC
AAR. Analyzed the data: RR CVR SJS. Contributed reagents/
materials/analysis tools: CVR SJS PSM KAL. Wrote the
manuscript: RR CVR SJS DCM FRR KMS RGG.
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